Synthesis of oligosaccharides

ABSTRACT

This invention relates to a method for the enzymatic synthesis of oligosaccharides, preferably human milk oligosaccharides (HMOs) The method comprises the enzymatic transfer of a glycosyl moiety and subsequent removal of by-products, such as lactose, by nanofiltration using a membrane comprising an active polyamide layer.

FIELD OF THE INVENTION

This invention relates to a method for the enzymatic synthesis ofoligosaccharides, preferably human milk oligosaccharides (HMOs).

BACKGROUND OF THE INVENTION

In recent years, the manufacture and commercialization of complexcarbohydrates including naturally secreted oligosaccharides haveincreased significantly due to their roles in numerous biologicalprocesses occurring in living organisms. Secreted oligosaccharides suchas human milk oligosaccharides (HMOs) are carbohydrates which havegained much interest in recent years and are becoming importantcommercial targets for nutrition and therapeutic industries. Inparticular, the synthesis of these HMOs has increased significantly dueto the role of HMOs in numerous biological processes occurring inhumans. The great importance of HMOs is directly linked to their uniquebiological activities such as antibacterial, antiviral, immune systemand cognitive development enhancing activities. Human milkoligosaccharides are found to act as prebiotics in the human intestinalsystem helping to develop and maintain the intestinal flora.Furthermore, they have also proved to be anti-inflammatory, andtherefore these compounds are attractive components in the nutritionalindustry for the production of, for example, infant formulas, infantcereals, clinical infant nutritional products, toddler formulas, or asdietary supplements or health functional food for children, adults,elderly or lactating women, both as synthetically composed and naturallyoccurring compounds and salts thereof. Likewise, the compounds are alsoof interest in the medicinal industry for the production of therapeuticsdue to their prognostic use as immunomodulators. However, the synthesesand purification of these oligosaccharides and their intermediatesremained a challenging task for science.

The availability of naturally occurring sialylated human milkoligosaccharides is limited from natural sources. Mature human milk isthe natural milk source that contains the highest concentrations of milkoligosaccharides (12-14 g/l), other milk sources are cow's milk (0.01g/l), goat's milk and milk from other mammals. Approximately 200 HMOshave been detected from human milk by means of combination of techniquesincluding microchip liquid chromatography mass spectrometry (HPLCChip/MS) and matrix-assisted laser desorption/ionization Fouriertransform ion cyclotron resonance mass spectrometry (MALDI-FT ICR MS)(Ninonuevo et al. J. Agric. Food Chem. 54, 7471 (2006)), from which todate at least 115 oligosaccharides have been structurally determined(Urashima et al.: Milk Oligosaccharides, Nova Medical Books, N Y, 2011;Chen Adv. Carbohydr. Chem. Biochem. 72, 113 (2015)). Due to the largenumber of similar HMOs and their low concentrations in mammalian milk,isolation of HMOs is a difficult task even in milligram quantities. Todate only analytical HPLC methodologies have been developed for theisolation of some HMOs from natural sources. It is therefore difficultto provide suitable HMO replacements in foods, particularly in infantformulae which display at least part of the entire spectrum of HMOs.

Biotechnological approaches have proved to be promising andcost-efficient for the synthesis of a variety of HMOs, especially tri-or tetrasaccharide HMOs. Precisely, those HMOs can be produced inaqueous media by fermentation of genetically modified bacteria, yeastsor other microorganisms. See, for example, WO 01/04341, WO 2007/101862,WO 2010/070104, WO 2010/142305, WO 2012/112777, WO 2014/153253, WO2015/036138, WO 2015/150328, WO 2016/008602, EP-A-2722394, Priem et al.Glycobiology 12, 235 (2002), Drouillard et al. Angew. Chem. Int. Ed. 45,1778 (2006), Han et al. Biotechnol. Adv. 30, 1268 (2012), Lee et al.Microb. Cell Fact. 11:48 (2012) and Baumgartner et al. Microb. CellFact. 12:40 (2013).

Efforts have also been made to develop processes for synthesizingenzymatically mixtures of HMO oligosaccharides, see e.g. EP-A-577580, WO2012/156897, WO 2012/156898, WO 2016/063326, WO 2016/157108 or WO2016/199071. Such processes have provided reaction mixtures containing aplurality of different oligosaccharides.

Aydo{hacek over (g)}an et al. (Separ. Sci. Technol. 33, 1767 (1998))stated that nanofiltration is not a very suitable method forfractionation of sugars.

WO 98/15581 discloses the retention characteristics of salts andcarbohydrates (lactose, sialyllactose, lacto-N-triose II,lacto-N-tetraose), and concludes that while both GE GH and GE GEpolyamide membranes allow ions to pass, the GE GE membrane retainssialyllactose or similar trisaccharides more efficiently than the GE GHmembrane. No conclusion about whether lactose could be separated fromhigher oligosaccharides was drawn.

Goulas et al. (J. Sci. Food Agric. 83, 675 (2003)) investigated thefractionating of commercial oligosaccharide mixtures by nanofiltrationand observed that the rejection and permeate concentration values givenby the membranes for the sugars during the filtration of single-sugarsolutions would be not the same as if these sugars had been in a mixedsolution.

WO 2005/067962 discloses the isolation of goat milk oligosaccharidescomprising filtration of skimmed goat milk ultrafiltration permeate witha ceramic membrane of 1-5 kDa. Although a partial separation of saltsand lactose is anticipated, the application is silent to quantify this.Nevertheless, the method further comprises active charcoalchromatography, ion exchange chromatography and electrodialysis toremove lactose and salts.

Luo et al. (Biores. Technol. 166, 9 (2014)) and Nordvang et al. (Separ.Purif Technol. 138, 77 (2014)) tested the separation of enzymaticallyproduced 3′-SL from lactose by nanofiltration; although apolyethersulphone (PES) membrane with a MWCO of 1000-1400 Da and asulphonated PES membrane with a MWCO of 600-800 Da were suitable toseparate the most of the lactose after diafiltration, the loss of 3′-SLwas significant and its purity after separation was rather moderate,thus 3′-SL was further purified with anion exchange chromatography.

Evidence is accumulating that the resident community of microbes, calledthe microbiome, in the human digestive tract plays a major role inhealth and disease. When the normal composition of the microbiome isthrown off balance, the human host can suffer consequences. Recentresearch has implicated microbiome imbalances in disorders as diverse ascancer, obesity, inflammatory bowel disease, psoriasis, asthma, andpossibly even autism. HMOs are believed to positively modulate themicrobiome, and they are of increasing interest for this purpose.However, the remarkable diversity of HMOs, coupled with their lack ofavailability, has hampered studies of the specific functions ofindividual HMOs. The prior art enzymatic reactions, although being ableto produce more diversified HMOs, suffer from the fact that theconversion rate is rather moderate and the mixture contains asubstantial amount of lactose.

Accordingly, there is a clear need for specific HMOs or combinations ofHMOs, without substantial amount of lactose, to modulate the microbiomein a desired manner, so as to address specific human health issues,and/or to produce those HMOs enzymatically in more efficient way.

SUMMARY OF THE INVENTION

The first aspect of the invention provides a method for producing acompound Gly-A by glycosylating acceptor A with a donor Gly-B under thecatalysis of a glycosidase capable of transferring the Gly moiety fromthe donor to the acceptor thereby forming a mixture containing Gly-A, A,Gly-B and B, comprising:

-   -   a) contacting said mixture with a polyamide nanofiltration        membrane with a molecular weight cut-off (MWCO) of 600-3500 Da        ensuring the retention of Gly-A, A and Gly-B and allowing at        least a part of compound B to pass, wherein the MgSO₄ rejection        of the membrane is 50-90%,    -   b) optional diafiltration of the retentate obtained in step a),    -   c) and collecting the retentate obtained in step a) or step b)        enriched in compound Gly-A,    -   wherein A means a tri- or higher oligosaccharide,    -   B means a disaccharide,    -   Gly means a glycosyl moiety,    -   Gly-A means compound A glycosylated with the Gly moiety, and    -   Gly-B means compound B glycosylated with the Gly moiety,    -   provided that compounds A and Gly-B are not identical.

The second aspect of the invention provides a method for improving theconversion of the product formation of compound Gly-A in an enzymaticreaction wherein acceptor A is glycosylated with a donor Gly-B under thecatalysis of a glycosidase capable of transferring the Gly moiety fromthe donor to the acceptor thereby forming a mixture containing Gly-A, A,Gly-B and B, comprising:

-   -   a) contacting said mixture, under diafiltration condition, with        a polyamide nanofiltration membrane with a molecular weight        cut-off (MWCO) of 600-3500 Da ensuring the retention of Gly-A, A        and Gly-B and allowing at least a part of compound B to pass,        wherein the MgSO₄ rejection of the membrane is 50-90%,    -   b) and collecting the retentate enriched in compound Gly-A,    -   wherein A means a tri- or higher oligosaccharide,    -   B means a disaccharide,    -   Gly means a glycosyl moiety,    -   Gly-A means compound A glycosylated with the Gly moiety, and    -   Gly-B means compound B glycosylated with the Gly moiety,    -   provided that compounds A and Gly-B are not identical.

In both aspects, compound B is preferably lactose.

In both aspects, also preferably, compound A is a tri- tooctasaccharide, more preferably a tri-, tetra- or pentasaccharide.

In both aspects, also preferably, the Gly residue is a monosaccharideresidue. More preferably, the monosaccharide Gly residue is fucosyl andthe glycosidase is a fucosidase, especially a transfucosidase, or theGly residue is sialyl and the glycosidase is a sialidase, especially atranssialidase.

In both aspects, also preferably, the polyamide nanofiltration membraneis a thin-film composite (TFC) membrane.

In both aspects, yet preferably, the polyamide nanofiltration membraneis a phenylene diamine or a piperazine membrane.

In a preferred embodiment in both aspects, the method comprisesdiafiltration.

DETAILED DESCRIPTION OF THE INVENTION Terms and Definitions

The term “monosaccharide” means a sugar of 5-9 carbon atoms that is analdose (e.g. D-glucose, D-galactose, D-mannose, D-ribose, D-arabinose,L-arabinose, D-xylose, etc.), a ketose (e.g. D-fructose, D-sorbose,D-tagatose, etc.), a deoxysugar (e.g. L-rhamnose, L-fucose, etc.), adeoxy-aminosugar (e.g. N-acetylglucosamine, N-acetylmannosamine,N-acetylgalactosamine, etc.), an uronic acid, a ketoaldonic acid (e.g.sialic acid) or equivalents.

The term “disaccharide” means a carbohydrate consisting of twomonosaccharide units linked to each other by an interglycosidic linkage.

The term “tri- or higher oligosaccharide” means a sugar polymerconsisting of at least three, preferably from three to eight, morepreferably from three to six, monosaccharide units (vide supra). Theoligosaccharide can have a linear or branched structure containingmonosaccharide units that are linked to each other by interglycosidiclinkages.

The term “human milk oligosaccharide” or “HMO” means a complexcarbohydrate found in human breast milk (Urashima et al.: MilkOligosaccharides, Nova Medical Books, NY, 2011; Chen Adv. Carbohydr.Chem. Biochem. 72, 113 (2015)). The HMOs have a core structure being alactose unit at the reducing end that is elongated by one or moreβ-N-acetyl-lactosaminyl and/or one or more β-lacto-N-biosyl units, andwhich core structures can be substituted by an α L-fucopyranosyl and/oran α-N-acetyl-neuraminyl (sialyl) moiety. In this regard, the non-acidic(or neutral) HMOs are devoid of a sialyl residue, and the acidic HMOshave at least one sialyl residue in their structure. The non-acidic (orneutral) HMOs can be fucosylated or non-fucosylated. Examples of suchneutral non-fucosylated HMOs include lacto-N-triose (LNTri,GlcNAc(β1-3)Gal(β1-4)Glc), lacto-N-tetraose (LNT), lacto-N-neotetraose(LNnT), lacto-N-neohexaose (LNnH), para-lacto-N-neohexaose (pLNnH),para-lacto-N-hexaose (pLNH) and lacto-N-hexaose (LNH). Examples ofneutral fucosylated HMOs include 2′-fucosyllactose (2′-FL),lacto-N-fucopentaose I (LNFP-I), lacto-N-difucohexaose I (LNDFH-I),3-fucosyllactose (3-FL), difucosyllactose (DFL), lacto-N-fucopentaose II(LNFP-II), lacto-N-fucopentaose III (LNFP-III), lacto-N-difucohexaoseIII (LNDFH-III), fucosyl-lacto-N-hexaose II (FLNH-II),lacto-N-fucopentaose V (LNFP-V), lacto-N-fucopentaose VI (LNFP-VI),lacto-N-difucohexaose II (LNDFH-II), fucosyl-lacto-N-hexaose I (FLNH-I),fucosyl-para-lacto-N-hexaose I (FpLNH-I),fucosyl-para-lacto-N-neohexaose II (F-pLNnH II) andfucosyl-lacto-N-neohexaose (FLNnH). Examples of acidic HMOs include3′-sialyllactose (3′-SL), 6′-sialyllactose (6′-SL),3-fucosyl-3′-sialyllactose (FSL), LST a, fucosyl-LST a (FLST a), LST b,fucosyl-LST b (FLST b), LST c, fucosyl-LST c (FLST c), sialyl-LNH(SLNH), sialyl-lacto-N-hexaose (SLNH), sialyl-lacto-N-neohexaose I(SLNH-I), sialyl-lacto-N-neohexaose II (SLNH-II) anddisialyl-lacto-N-tetraose (DSLNT).

The term “sialyl” or “sialyl moiety” means the glycosyl residue ofsialic acid (N-acetyl-neuraminic acid, Neu5Ac), preferably linked withα-linkage:

The term “fucosyl” means an L-fucopyranosyl group, preferably linkedwith α-interglycosidic linkage:

“N-acetyl-glucosaminyl” means anN-acetyl-2-amino-2-deoxy-D-glucopyranosyl (GlcNAc) group, preferablylinked with β-linkage:

“N-acetyl-lactosaminyl” means the glycosyl residue ofN-acetyl-lactosamine (LacNAc, Galpβ1-4GlcNAcp), preferably linked withβ-linkage:

Furthermore, the term “lacto-N-biosyl” means the glycosyl residue oflacto-N-biose (LNB, Galpβ1-3GlcNAcp), preferably linked with β-linkage:

Rejection factor of a salt (in percent) is calculated as(1−κ_(p)/κ_(r))·100, wherein κ_(p) is the conductivity of the salt inthe permeate and Kr is the conductivity of the salt in the retentate.The retentate concentration is practically equal to the feedconcentration concerning the salt. The procedure for measuring rejectionof salts is disclosed in the working examples below.

Rejection factor of a carbohydrate (in percent) is calculated as(1−C_(p)/C_(r))·100, wherein C_(p) is the concentration of thecarbohydrate in the permeate and C_(r) is the concentration of thecarbohydrate in the retentate. The retentate concentration ispractically equal to the feed concentration concerning the carbohydrate.The procedure for measuring rejection of a carbohydrate is disclosed inthe examples.

Separation factor concerning two carbohydrates is calculated as(C_(p1)/C_(r1))/(C_(p2)/C_(r2)), wherein C_(p1) and C_(p2) are theconcentrations of the first and the second carbohydrate, respectively,in the permeate, and C_(r1) and C_(r2) are the concentrations of thefirst and the second carbohydrate, respectively, in the retentate.

“Pure water flux” is defined as the volume of purified water (e.g.distilled water, RO water) that passes through a membrane per unit time,per unit area and per unit of transmembrane pressure under specifiedconditions (at 23-25° C., 10 bar and constant cross-flow of 300 l/h).The procedure for measuring the pure water flux is disclosed in example3 below.

Enzymatic Production of Gly-A

In the enzymatic reactions comprising the utilization of a glycosidase,a glycosyl moiety from a donor molecule is transferred to an acceptor,thereby forming a glycosylated acceptor and the residue of the donordevoid of the glycosyl moiety.

The enzymatic reaction comprising A as acceptor and Gly-B as donor underthe catalysis of a glycosidase that is able to transfer the glycosylresidue Gly from the donor to the acceptor, can be depicted as follows:

wherein A is a tri- or higher oligosaccharide, Gly is a monosaccharideglycosyl moiety, B is a disaccharide, Gly-B is disaccharide glycosylatedwith the Gly moiety and Gly-A is compound A glycosylated with the Glymoiety. However, the glycosidases, in general, are able to transfer theGly residue from the newly formed Gly-A back to the compound B that haspreviously been produced from Gly-B, therefore reaching an equilibrium:A+Gly-B⇄Gly-A+B. In these reactions the conversion rate is rathermoderate, meaning the valuable product Gly-A is comprised in thereaction mixture along with A, B and Gly-B.

In order to shift the equilibrium towards the product (Gly-A) formation,one may add any of A and Gly-B in excess, however the reaction mixturethus will contain that particular component in a significant amount, theseparation of which from the valuable product Gly-A may be problematicor cumbersome.

Another way to push the equilibrium for formation Gly-A is to(continuously) remove the other product, namely component B, from thereaction mixture.

The present inventors have surprisingly found that component B can beselectively separated (removed) from an aqueous mixture comprising A, B,Gly-A and Gly-B by a nanofiltration step, therefore the conversion rateis significantly increased and the reaction equilibrium is shifted forthe product formation of Gly-A so that mixture contains Gly-A inmajority.

Accordingly, in a first aspect, a method is provided for producing acompound Gly-A by glycosylating acceptor A with a donor Gly-B under thecatalysis of a glycosidase capable of transferring the Gly moiety fromthe donor to the acceptor thereby forming a mixture containing Gly-A, A,Gly-B and B, comprising:

-   -   a) contacting said mixture with a nanofiltration membrane with a        molecular weight cut-off (MWCO) of 600-3500 Da ensuring the        retention of Gly-A, A and Gly-B and allowing at least a part of        compound B to pass, wherein the active (top) layer of the        membrane is composed of polyamide, and wherein the MgSO₄        rejection of the membrane is 50-90%,    -   b) optional diafiltration of the retentate obtained in step a),    -   c) and collecting the retentate obtained in step a) or step b)        enriched in compound Gly-A,    -   wherein A means a tri- or higher oligosaccharide,    -   B means a disaccharide,    -   Gly means a glycosyl moiety,    -   Gly-A means compound A glycosylated with the Gly moiety, and    -   Gly-B means compound B glycosylated with the Gly moiety,    -   provided that compound A and Gly-B are not identical.

In a second aspect, a method is provided for improving the conversion ofthe product formation of compound Gly-A in an enzymatic reaction whereinacceptor A is glycosylated with a donor Gly-B under the catalysis of aglycosidase capable of transferring the Gly moiety from the donor to theacceptor thereby forming a mixture containing Gly-A, A, Gly-B and B,comprising:

-   -   a) contacting said mixture, under diafiltration condition, with        a nanofiltration membrane with a molecular weight cut-off (MWCO)        of 600-3500 Da ensuring the retention of Gly-A, A and Gly-B and        allowing at least a part of compound B to pass, wherein the        active (top) layer of the membrane is composed of polyamide, and        wherein the MgSO₄ rejection of the membrane is 50-90%,    -   b) and collecting the retentate enriched in compound Gly-A,    -   wherein A means a tri- or higher oligosaccharide,    -   B means a disaccharide,    -   Gly means a glycosyl moiety,    -   Gly-A means compound A glycosylated with the Gly moiety, and    -   Gly-B means compound B glycosylated with the Gly moiety,    -   provided that compound A and Gly-B are not identical.

The term “ensuring the retention of Gly-A, A and Gly-B” preferably meansthat, during the nanofiltration step, Gly-A, A and Gly-B do not pass, orat least significantly do not pass, through the membrane and thus theirvast majority will be present in the retentate. The term “allowing atleast a part of compound B to pass through the membrane” preferablymeans, that compound B, at least partially, can penetrate the membraneand be collected in the permeate. In the first aspect of the invention,in case of high rejection (about 90%) of compound B, a subsequentdiafiltration with pure water may be necessary to bring all or at leastthe majority of compound B in the permeate. The higher the rejection ofcompound B the more diafiltration water is necessary for efficientseparation.

The term “contacting said mixture, under diafiltration condition, with ananofiltration membrane” preferably means that the nanofiltration iscarried out so that water is continuously added to the retentate andpassed through the membrane. In doing so, all or at least the majorityof compound B can be removed from the retentate and brought to thepermeate.

The applied nanofiltration membrane shall be tight for Gly-A, A andGly-B in order that they are efficiently retained. Preferably, therejection of Gly-A, A and Gly-B is more than 95%, more preferably 97%,even more preferably 99%. Membranes with MWCO of more than 3500 Da areexpected to allow more or significant amount of Gly-A, A and Gly-B passthrough the membrane thus show a reduced retention of Gly-A, A and Gly-Band therefore are not suitable for the purposes of the invention, andcan be excluded. In the same time, membranes with MWCO of less than 600Da can also be excluded, because—together with the retention of Gly-A, Aand Gly-B—that of the mono- and disaccharides is also expected, meaningthat the overall separation of the compounds would likely be poor. Inthis regard, it is preferred that the rejection of the disaccharide(compound B) is not more than 80-90%. If the disaccharide rejectionturns to be 90±1-2%, the rejection of at least Gly-A shall preferably bearound 99% or higher in order to achieve a practically satisfyingseparation.

It has been found that the above requirements are simultaneouslyfulfilled when the membrane is relatively loose for MgSO₄, that is itsrejection is about 50-90%. In this regard the above specified membraneis tight for Gly-A, A and Gly-B, and loose for mono- and disaccharides,and as well as for MgSO₄. Therefore, it is possible to separate e.g.lactose, which is a precursor in making human milk oligosaccharidesenzymatically or by fermentation, from the human milk oligosaccharidesproduct by nanofiltration with a good efficacy, and additionally asubstantial part of divalent ions also passes to the permeate. In someembodiments, the MgSO₄ rejection factor is 60-90%, 70-90%, 50-80%,50-70%, 60-70% or 70-80%. Preferably, the MgSO₄ rejection factor on saidmembrane is 80-90%.

Also preferably, the membrane has a rejection factor for NaCl that islower than that for MgSO₄. In one embodiment, the rejection factor forNaCl is not more than 50%. In other embodiment, the rejection factor forNaCl is not more than 40%. In other embodiment, the rejection factor forNaCl is not more than 30%. In this latter embodiment, a substantialreduction of all monovalent salts in the retentate is also achievable.

Also preferably, the membrane has a rejection factor for NaCl that islower than that for MgSO₄. In one embodiment, the rejection factor forNaCl is not more than 50%. In other embodiment, the rejection factor forNaCl is not more than 40%. In other embodiment, the rejection factor forNaCl is not more than 30%. In this latter embodiment, a substantialreduction of all monovalent salts in the retentate is also achievable.

Also preferably, in some embodiments, the pure water flux of themembrane is at least 50 l/m²h. Preferably, the pure water flux of themembrane is at least 60 l/m²h, at least 70 l/m²h, at least 80 l/m²h orat least 90 l/m²h.

The active or the top layer of nanofiltration membrane suitable for thepurpose of the invention is preferably made of polyamide. Althoughmembranes of different type seem to have promising separation efficacy,for example NTR-7450 having sulphonated PES as active layer forseparating lactose and 3′-SL (Luo et al. (Biores. Technol. 166, 9(2014); Nordvang et al. (Separ. Purif Technol. 138, 77 (2014)), theabove specified membrane used in the invention shows always betterseparation of lactose from an HMO. In addition, the above mentionedNTR-7450 membrane is subject to fouling, which typically results in adrop in flux, increasing the lactose rejection and therefore a reducedseparation factor (see examples).

Yet preferably, the polyamide membrane is a polyamide with phenylenediamine or piperazine building blocks as amine, more preferablypiperazine (referred to as piperazine-based polyamide, too).

Yet preferably, the membrane suitable for the purpose of the presentinvention is a thin-film composite (TFC) membrane.

An example of suitable piperazine based polyamide TFC membranes isTriSep® UA60.

The claimed method applies a nanofiltration membrane characterized bysome or all of the above features and thus one or more of the followingbenefits are provided: selectively and efficiently removes compound B, adisaccharide, preferably lactose, from compounds Gly-A, A and Gly-Bwhich are tri- or higher oligosaccharides, preferably HMOs, therebyyielding an enriched Gly-A, A and Gly-B fraction and/or improvingconversion of Gly-A; removes efficiently monovalent as well as divalentsalts therefore no ion exchange step is necessary or, if desalination isstill needed, the ion exchange treatment requires substantially lessresin; higher flux during the nanofiltration can be maintained comparedto other membranes used for the same or similar purpose in the priorart, which reduces the operation time; the membrane applied in theclaimed method is less prone to getting clogged compared to the priorart solutions; the membrane applied in the claimed can be cleaned andregenerated completely therefore can be recycled without substantialreduction of its performance.

The nanofiltration membrane defined in the method of the invention ismore beneficial compared to the prior art membranes used for the same orsimilar purpose as that of the present invention. Specifically,polyvinylidene fluoride (PVDF) membrane of Luo et al. or Nordvang et al.(ETNA01PP, MWCO: 1000 Da, Alfa Laval) rejects tri- to hexasaccharidesless efficiently and the separation factor over lactose is substantiallylower; sulphonated PES membrane of Luo et al. or Nordvang et al.(NTR-7450, MWCO: 600-800, Nitto-Denko), besides showing lower separationfactor of tri- to hexasaccharides over lactose, gets easily clogged; GEGE (polyamide, MWCO: 1000 Da) and GE GH (polyamide, MWCO: 2500 Da)membranes of WO 98/15581, besides showing lower separation factor oftri- to hexasaccharides over lactose, operate at lower flux and retainhigher amount of salts in the permeate due to high NaCl rejectionfactor.

Accordingly, in one embodiment of the first aspect of the invention, amethod is provided for producing a compound Gly-A by glycosylatingacceptor A with a donor Gly-B under the catalysis of a glycosidasecapable of transferring the Gly moiety from the donor to the acceptorthereby forming a mixture containing Gly-A, A, Gly-B and B, comprising:

-   -   a) contacting said mixture with a piperazine-based polyamide        nanofiltration membrane with a molecular weight cut-off (MWCO)        of 1000-3500 Da ensuring the retention of Gly-A, A and Gly-B and        allowing at least a part of compound B to pass through the        membrane, wherein the MgSO₄ rejection factor on said membrane is        80-90%, and wherein        -   the NaCl rejection factor on said membrane is lower than            that for MgSO₄, and/or        -   the pure water flux value of said membrane is at least 50            l/m²h,    -   b) a subsequent optional diafiltration of the retentate obtained        in step a),    -   c) and collecting the retentate obtained in step a) or step b)        enriched in compound Gly-A.

In one embodiment of the second aspect, a method is provided forimproving the conversion of the product formation of compound Gly-A inan enzymatic reaction wherein acceptor A is glycosylated with a donorGly-B under the catalysis of a glycosidase capable of transferring theGly moiety from the donor to the acceptor thereby forming a mixturecontaining Gly-A, A, Gly-B and B, comprising:

-   -   a) contacting said mixture, under diafiltration condition, with        a nanofiltration membrane with a molecular weight cut-off (MWCO)        of 1000-3500 Da ensuring the retention of Gly-A, A and Gly-B and        allowing at least a part of compound B to pass, wherein the        active (top) layer of the membrane is composed of polyamide, and        wherein the MgSO₄ rejection of the membrane is 80-90%, and        wherein        -   the NaCl rejection factor on said membrane is lower than            that for MgSO₄, and/or        -   the pure water flux value of said membrane is at least 50            l/m²h,    -   b) and collecting the retentate enriched in compound Gly-A.

Preferably, in the above embodiments, the NaCl rejection factor of themembrane is at most the half of the MgSO₄ rejection factor.

To achieve all the benefits mentioned above, the nanofiltration membraneto be applied in both aspects of the invention, preferably:

-   -   is a piperazine-based polyamide membrane with a MWCO of        1000-3500 Da,    -   has a MgSO₄ rejection of 50-90%, preferably 80-90%,    -   has a NaCl rejection of not more than 30%, and    -   has a pure water flux value of at least 50 l/m²h, preferably 90        l/m²h.

Improvement of the conversion of the enzymatic synthesis means that themolar ratio of Gly-A is increased compared to that in the equilibriumbefore the selective removal of disaccharide B takes place. In anequilibrium A+Gly-B

Gly-A+B, the conversion of Gly-A is calculated as the fraction of themolar concentration of Gly-A and that of the acceptor A or donor Gly-Bin percent depending on which one was added to the reaction mixture inlower amount. The method of the invention increases the conversion,compared to that in the equilibrium before removal of compound B, by atleast 10%, preferably by at least 25%, more preferably by at least 50%,even more preferably by at least 75%, particularly by at least 100%.

Also in a preferred embodiment, compound B is lactose and compound Gly-B(serving as donor in the above disclosed enzymatic reaction) is aglycosylated lactose wherein the Gly residue is attached to the lactoseby an interglycosidic linkage. The Gly residue is preferably amonosaccharide residue, therefore Gly-B is a trisaccharide.

Also preferably, compound A (serving as acceptor in the above disclosedenzymatic reaction) is a tri- or higher oligosaccharide, more preferablytri- to hexasaccharides. Accordingly, Gly-A (being as the intendedproduct in the above disclosed enzymatic reaction) is a glycosylatedcompound A, that is a glycosylated tri- or higher oligosaccharide. In afurther preferred embodiment, the Gly residue is a monosaccharideresidue, therefore Gly-A is a tetra- or higher oligosaccharide, morepreferably tetra- to heptasaccharide.

In the enzymatic reaction disclosed above an enzyme capable oftransferring the Gly residue from Gly-B to compound A is utilized. Suchan enzyme comprises a glycosidase activity, preferably atransglycosidase activity thereby transferring a glycosyl moiety (e.g. asialyl moiety, a fucosyl moiety, an N-acetyllactosaminyl moiety, alacto-N-biosyl moiety, etc.) and forming a new bond between thetransferred glycosyl moiety and the acceptor molecule (compound A) at aspecific position of the acceptor.

According to a more preferred embodiment, compound B is lactose and Glyis

-   -   fucosyl (thus Gly-B is a fucosyllactose, particularly 2′-FL or        3-FL),    -   sialyl (thus Gly-B is a sialyllactose, particularly 3′-SL or        6′-SL),    -   N-acetylglucosaminyl (thus Gly-B is a        N-acetylglucosaminyllactose, particularly lacto-N-triose II),    -   lacto-N-biosyl (thus Gly-B is a lacto-N-biosyl lactose,        particularly LNT), or    -   N-acetyllactosaminyl (thus Gly-B is a N-acetyllactosaminyl        lactose, particularly LNnT);        and a suitable glycosidase is a fucosidase (when Gly is        fucosyl), a sialidase (when Gly is sialyl), a        N-acetylhexosaminidase (when Gly is N-acetylglucosaminyl), a        lacto-N-biosidase (when Gly is lacto-N-biosyl) or        N-acetyllactosaminidase (when Gly is N-acetyllactosaminyl).

Also more preferably, compound A is characterized by formula 1 orformula 2:

-   -   wherein R₁ is fucosyl or H,    -   R₂ is selected from N-acetyl-lactosaminyl and lacto-N-biosyl        groups, wherein the N-acetyl lactosaminyl group may carry a        glycosyl residue comprising one or more N-acetyl-lactosaminyl        and/or one or more lacto-N-biosyl groups; any        N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue,    -   R₃ is H or N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one or more        N-acetyl-lactosaminyl and/or one or more lacto-N-biosyl groups;        any N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue, and    -   each R₄ independently is sialyl or H,    -   with the proviso that at least one of R₁ or R₄ is not H;        and within the scope of formula 1, compound A can be        characterized by formula 1a or formula 1b:

-   -   wherein R₁ is as defined above,    -   R_(2a) is an N-acetyl-lactosaminyl group optionally substituted        with a glycosyl residue comprising one N-acetyl-lactosaminyl        and/or one lacto-N-biosyl group; any N-acetyl-lactosaminyl and        lacto-N-biosyl group can be substituted with one or more sialyl        and/or fucosyl residue, but preferably void of a sialyl and/or        fucosyl residue,    -   R_(3a) is H or an N-acetyl-lactosaminyl group optionally        substituted with a lacto-N-biosyl group; any        N-acetyl-lactosaminyl and lacto-N-biosyl group can be        substituted with one or more sialyl and/or fucosyl residue, but        preferably void of a sialyl and/or fucosyl residue,    -   R_(2b) is a lacto-N-biosyl group optionally substituted with        sialyl and/or fucosyl residue, but preferably void of a sialyl        and/or fucosyl residue, and    -   R_(3b) is H or an N-acetyl-lactosaminyl group optionally        substituted with one or two N-acetyl-lactosaminyl and/or one        lacto-N-biosyl group; any N-acetyl-lactosaminyl and        lacto-N-biosyl group can be substituted with one or more sialyl        and/or fucosyl residue, but preferably without a sialyl and/or        fucosyl residue;        and preferably, the compounds of formulae 1a and 1b have one or        more of the following linkages and modifications:    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(2a) in formula 1a is attached to another        N-acetyl-lactosaminyl group by a 1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(2a) in        formula 1a is attached to the N-acetyl-lactosaminyl group by a        1-3 interglycosidic linkage,    -   the lacto-N-biosyl group in the glycosyl residue of R_(3a) in        formula 1a is attached to the N-acetyl-lactosaminyl group by a        1-3 interglycosidic linkage,    -   the N-acetyl-lactosaminyl group in the glycosyl residue of        R_(3b) in formula 1b is attached to another        N-acetyl-lactosaminyl group by a 1-3 or 1-6 interglycosidic        linkage, and    -   the lacto-N-biosyl group in the glycosyl residue of R_(3b) in        formula 1b is attached to the N-acetyl-lactosaminyl group by a        1-3 interglycosidic linkage.

Preferably, compound A defined above is further characterized in that:

-   -   if present, the fucosyl residue attached to the        N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked        to        -   the galactose of the lacto-N-biosyl group with a 1-2            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            1-4 interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the N-acetyl-lactosaminyl group            with a 1-3 interglycosidic linkage,    -   if present, the sialyl residue attached to the        N-acetyl-lactosaminyl and/or the lacto-N-biosyl group is linked        to        -   the galactose of the lacto-N-biosyl group with a 2-3            interglycosidic linkage and/or        -   the N-acetyl-glucosamine of the lacto-N-biosyl group with a            2-6 interglycosidic linkage and/or        -   the galactose of the N-acetyl-lactosaminyl group with a 2-6            interglycosidic linkage.

According to a further preferred embodiments of compound A, a compoundaccording to general subformulae 1a, 1b or 2 may be:

-   -   a trisaccharide such as 2′-O-fucosyllactose (2′-FL),        3-O-fucosyllactose (3-FL), 3′-O-sialyllactose (3′-SL),    -   a tetrasaccharide such as lacto-N-tetraose (LNT),        lacto-N-neotetraose (LNnT),    -   a pentasaccharide such as lacto-N-fucopentaose I (LNFP-I,        Fucα1-2Galβ1-3GlcNAcβ1-3Galβ1-4Glc),        Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc, lacto-N-fucopentaose II        (LNFP-II, Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4Glc),        lacto-N-fucopentaose III (LNFP-III,        Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc), lacto-N-fucopentaose V        (LNFP-V, Galβ1-3GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc),        lacto-N-fucopentaose VI (LNFP-VI,        Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc),        Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc, LST a        (NeuAcα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc), LST b        (Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc), LST c        (NeuAcα2-6Galβ1-4GlcNAcβ1-3Galβ1-4Glc),        Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc,    -   a hexasaccharide such as lacto-N-hexaose (LNH,        Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        lacto-N-neohexaose (LNnH,        Galβ1-4GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        para-lacto-N-hexaose (pLNH,        Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc),        para-lacto-N-neohexaose (pLNnH,        Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), disialyl-LNT        (DSLNT, NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3Galβ1-4Glc), LNDFH        II (Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc),        Galβ1-3[Neu5Acα2-6][Fucα1-4]GlcNAcβ1-3Galβ1-4Glc,        Galβ1-3[Neu5Acα2-6]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc, LNDFH III        (Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]Glc,    -   a heptasaccharide such as fucosyl-LNH I (FLNH-I,        Fucα1-2Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        fucosyl-LNH II (FLNH-II,        Galβ1-4[Fucα1-3]GlcNAcβ1-6[Galβ1-3GlcNAcβ1-3]Galβ1-4Glc),        fucosyl-para-LNH I (FpLNH-I,        Galβ1-3GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc),        fucosyl-para-LNH II (FpLNH-II,        Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc),        Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc,        Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc,        sialyl-LNH (SLNH,        Galβ1-3GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        sialyl-LNnH I (SLNnH-I,        Galβ1-4GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        sialyl-LNnH II (SLNnH-II,        Galβ1-4GlcNAcβ1-6[NeuAcα2-6Galβ1-4GlcNAcβ1-3]Galβ1-4Glc),        Fucα1-2Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,        Galβ1-3[Fucα1-4]GlcNAcβ1-3[Galβ13-4GlcNAcβ1-6]Galβ1-4Glc,        Galβ1-4[Fucα1-3]GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc,        NeuAcα2-3Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,    -   an octasaccharide such as difucosyl-LNH I (DFLNH-I,        Galβ1-4[Fucα1-3]GlcNAcβ1-6[Fucα1-2Galβ11-3GlcNAcβ1-3]Galβ1-4Glc),        difucosyl-para-LNH (DFpLNH,        Galβ1-3[Fucα1-4]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc),        difucosyl-para-LNnH (DFpLNnH,        Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc),        lacto-N-octaose (LNO,        Galβ1-3GlcNAcβ1-3[Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        lacto-N-neooctaose (LNnO,        Galβ1-4GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        iso-lacto-N-octaose (iLNO,        Galβ1-3GlcNAcβ1-3[Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        para-lacto-N-octaose (pLNO,        Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc),        fucosyl-sialyl-LNH (FSLNH,        NeuAcα2-3Galβ1-3GlcNAcβ1-3[Galβ1-4[Fucα1-3]GlcNAcβ1-6]Galβ1-4Glc),        fucosyl-sialyl-LNH II (FSLNH-II,        Fucα1-2Galβ1-3GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc),        disialyl-LNH I (DSLNH-I,        NeuAcα2-6Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3GlcNAcβ1-3]Galβ1-4Glc),        disialyl-LNH II (DSLNH-II,        Galβ1-4GlcNAcβ1-6[NeuAcα2-3Galβ1-3[NeuAcα2-6]GlcNAcβ1-3]Galβ1-4Glc),        disialyl-LNnH (DSLNnH,        NeuAcα2-6Galβ1-4GlcNAcβ1-6[NeuAcα2-6Galβ1-4GlcNAcβ1-3]Galβ1-4Glc),        Fucα1-2Galβ1-3[Fucα1-4]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,        Fucα1-2Galβ1-4[Fucα1-3]GlcNAcβ1-6[Galβ1-4GlcNAcβ1-3]Galβ1-4Glc,        NeuAcα2-3Galβ1-3[Fucα1-4]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,        NeuAcα2-6Galβ1-4[Fucα1-3]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,        Galβ1-4[Fucα1-3]GlcNAcβ1-3[NeuAcα2-6Galβ1-4GlcNAcβ1-6]Galβ1-4Glc.

In one embodiment, the glycosidase is a fucosidase. Fucosidases(classified in EC 3.2.1.38 and 3.2.1.51) are widespread in livingorganisms such as mammals, plants, fungi and bacteria. These enzymesbelong to the families 29, 35 and 95 of the glycoside hydrolases (GH29,GH35 and GH95) as defined by the CAZY nomenclature (http://www.cazy.org;Cantarel et al. Nucleic Acids Res. 37, D233 (2009)). Fucosidases fromGH29 are retaining enzymes (3D structure: (β/α)₈) whereas fucosidasesfrom GH95 are inverting enzymes (3D structure: (α/α)₆). The substratespecificity of the GH29 family is broad whereas that of the GH95 familyis strict to α1,2-linked fucosyl residues. The GH29 family seems to bedivided into two subfamilies. One subfamily typically has strictspecificity towards α1,3- and α1,4-fucosidic linkages. The members of afurther subfamily have broader specificity, covering all α-fucosyllinkages. Fucosidases generally hydrolyse the terminal fucosyl residuefrom glycans. However these enzymes are able to act as catalyst forfucosylation reaction due to their transfucosylation activity underkinetically controlled conditions.

The utility of glycosidases, including fucosidases, has benefited fromvarious engineering techniques.

In the technique of “rational engineering”, novel altered enzymes(mutants) are created by point mutation. The mutation generally affectsthe active site of the enzyme. Replacement of a catalytic nucleophilicresidue with a non-nucleophilic residue results in the formation of aninactive mutant or an altered enzyme with reduced transglycosylationactivity due the lack of appropriate environment for the formation ofthe reactive host-guest complex for transglycosylation. However, in thepresence of a more active fucosyl donor than the natural one, themutated enzyme is able to transfer efficiently the fucose residue to asuitable acceptor. Such a mutant glycosidase is termed glycosynthase.Rational engineering of enzymes generally requires reliance on thestatic 3D protein structure. By means of rational engineering, anα-1,2-L-fucosynthase from Bifidobacterium bifidum and efficientα-L-fucosynthases from Sulfolobus solfataricus and Thermotoga maritimawith acceptor dependent regioselectivity have recently been developedand provided [Wada et al. FEBS Lett. 582, 3739 (2008), Cobucci-Ponzanoet al. Chem. Biol. 16, 1097 (2009)]. These altered enzymes are devoid ofproduct hydrolysis activity.

A second technique of “directed evolution” involves random mutagenesisof a selected natural glycosidase enzyme to create a library of enzymevariants, each of which is altered in a single position or in multiplepositions. The variants can be inserted into suitable microorganismssuch as E. coli or S. cerevisiae for producing recombinant variants withslightly altered properties. Clones expressing improved enzyme variantsare then identified with a fast and reliable screening method, selectedand brought into a next round of mutation process. The recursive cyclesof mutation, recombination and selection are continued until mutant(s)with the desired activity and/or specificity is/are evolved. Anα-L-fucosidase from Thermotoga maritima has recently been converted intoan efficient α-L-transfucosidase by directed evolution [G. Osanjo et al.Biochemistry 46, 1022 (2007)]. The cited article describes the cloning,mutation, screening, recombination and protein purification steps indetail.

It is envisaged that transfucosidase and/or fucosynthase enzyme mutantsretaining transfucosidase activity and having a sequencesimilarity/homology to the sequence of the known and published enzymesequences, such as that of the α-L-transfucosidase of G. Osanjo et al,of at least 70%, such as 75%, preferably 80%, such as 85% can be used inthe present invention. Preferably, the sequence similarity is at least90%, more preferably 95%, 97%, 98% or most preferably 99%.

Engineered transfucosidases and fucosynthases possess a broader donorand acceptor specificity than the wild types of fucosidases andfucosyltransferases and so can be used in a particularly wide variety ofreactions. The engineered enzymes are, therefore, more advantageous forindustrial use.

In one particular embodiment, the fucosidase is α1,3/4-fucosidase or aα1,3/4-transfucosidase, that is those wild type or engineeredfucosidases that are able to transfer a fucose residue to the 3-positionof the glucose in an acceptor of formula 2, to the 3-position of theN-acetyl-glucosamine in a, preferably terminal, N-acetyl-lactosaminylgroup in an acceptor of formula 1, 1a or 1b, or to the 4-position of theN-acetyl-glucosamine in a, preferably terminal, lacto-N-biosyl group, inan acceptor of formula 1, 1a or 1b. In this regard, the preferredcompounds of Gly-A obtainable by α1,3/4-fucosylation of a suitableacceptor are DFL, SFL, LNFP-II, LNFP-III, LNDFH-I, LNDFH-II, LNDFH-III,FLNH-II, Galβ1-4[Fucα1-3]GlcNAcβ1-3[Galβ1-4GlcNAcβ1-6]Galβ1-4Glc,Galβ1-4GlcNAcβ1-3[Galβ1-4[Fucα1-3]GlcNAcβ1-6]Galβ1-4Glc, FpLNH-I,FpLNH-II, DFpLNH, Galβ1-4GlcNAcβ1-3Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4Glc,Galβ1-4[Fucα1-3]GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc, DFpLNH, DFpLNH,DFpLNnH, DFpLNnH, DFLNH-I, DFLNH-II, DFLNnH, DFLNnH, TFLNH, TFpLNnH,TFpLNH-II, FLST a, FLST c, FSLNH-III, FSLNnH-I, DFSLNH-I, DFSLNH-III,FDSLNT-I, FDSLNH-II, FDSLNH-III and FDSLNnH,

The α1,3/4-transfucosidase is preferably selected from α-L-fucosidasesas classified according to EC 3.2.1.111, having transfucosidaseactivity, such as the α1,3/4 fucosidase from Bifidobacterium longumsubsp. infantis ATCC 15697 as set forth in U.S. Pat. No. 8,361,756 asprotein of SEQ ID No. 18 and other fucosidases which have at least 60%,preferably at least 70%, more preferably at least 80%, particularly atleast 90%, identity with amino acid positions 56 to 345 of the α1,3/4fucosidase from Bifidobacterium longum subsp. infantis ATCC 15697.Examples of such other fucosidases are listed below in Table 1.

TABLE 1 Description Accession No. α-L-fucosidase KEY30716.1[Bifidobacterium longum subsp. infantis EK3] α-L-fucosidase[Bifidobacterium longum] WP_013140205.1 putative α1,3/4 fucosidaseKFI63931.1 [Bifidobacterium kashiwanohense JCM 15439] putative α1,3/4fucosidase KFI94501.1 [Bifidobacterium scardovii] α-L-fucosidase[Gardnerella vaginalis] WP_004574432.1 α-L-fucosidase [Gardnerellavaginalis] WP_019261748.1 hypothetical protein [Gardnerella vaginalis]WP_020759655.1 α-L-fucosidase [Gardnerella vaginalis] WP_009993891.1α-L-fucosidase [Gardnerella vaginalis] WP_004573610.1 α-L-fucosidase[Gardnerella vaginalis] WP_004120276.1 α-L-fucosidase [Gardnerellavaginalis] WP_004114072.1 α-L-fucosidase [Gardnerella vaginalis]WP_004137675.1 α-L-fucosidase [Gardnerella vaginalis] WP_014554869.1α-L-fucosidase [Bifidobacterium bifidum CAG:234] WP_022173522.1α-L-fucosidase [Actinomyces sp. ICM47] WP_009647833.1 hypotheticalprotein [Streptomyces ipomoeae] WP_009295550.1 α-L-fucosidase[Actinomyces sp. oral taxon 180] WP_009211856.1 hypothetical proteinWP_021611755.1 [Actinomyces sp. oral taxon 172] α-L-fucosidase[Bifidobacterium sp. 7101] WP_029678277.1 hypothetical protein[Actinomyces sp. HPA0247] WP_016461038.1 α-L-fucosidase [Actinomyces sp.ICM54] EWC96238.1 α-L-fucosidase [Actinomyces odontolyticus]WP_003795385.1 α-L-fucosidase [Atopobium sp. ICM58] WP_009055210.1α-L-fucosidase [Paenibacillus sp. J14] WP_028538247.1 α-L-fucosidase[Actinomyces odontolyticus] WP_003792781.1 α1,3/4 fucosidaseWP_015071771.1 [Propionibacterium acidipropionici] α-L-fucosidase[Propionibacterium acidipropionici] WP_028700846.1 hypothetical protein[Paenibacillus barengoltzii] WP_016312877.1 α-L-fucosidase [Actinomycessp. ICM39] WP_007588699.1 α-L-fucosidase [Propionibacterium jensenii]WP_028703334.1 α-L-fucosidase [Lactobacillus shenzhenensis]WP_022529554.1 hypothetical protein [Paenibacillus sp. HW567]WP_019912449.1 putative α-1 3/4-fucosidase WP_022032399.1 [Clostridiumhathewayi CAG:224] α-fucosidase [Clostridium hathewayi] WP_006775425.1α-L-fucosidase [Janibacter sp. HTCC2649] WP_009776262.1 α-fucosidase[Clostridium phytofermentans] WP_012201036.1 α-L-fucosidase[Enterococcus gallinarum] WP_029486307.1 uncharacterized protein[Blautia sp. CAG:237] WP_022215646.1 MULTISPECIES: α-L-fucosidase[Enterococcus] WP_005470131.1 α-L-fucosidase [Enterococcus gallinarumEG2] EEV33648.1 α-L-fucosidase [Ruminococcus sp. CAG:60] CCY33010.1α-L-fucosidase [Ruminococcus sp. CAG:9] WP_022380664.1 α-fucosidase[Blautia wexlerae] WP_025580031.1 α-fucosidase [Ruminococcus sp.5_1_39BFAA] WP_008706707.1 α-fucosidase [Paenibacillus sp. HGF5]WP_009593620.1 α-L-fucosidase [Paenibacillus sp. FSL H8-457] ETT68114.1hypothetical protein [Clostridium hathewayi] WP_002604401.1 hypotheticalprotein WP_017691196.1 [Paenibacillus sp. PAMC 26794] α-L-fucosidase[Paenibacillus sp. FSL R5-192] ETT29638.1 α-fucosidase [Paenibacillussp. Y412MC10] WP_015736742.1 α-L-fucosidase [Paenibacillus alvei]WP_021262981.1 α-fucosidase [Paenibacillus sp. UNC217MF] WP_028532504.1α-fucosidase [Paenibacillus alvei] WP_005546194.1 α-L-fucosidase[Paenibacillus alvei] WP_021254840.1 hypothetical protein [Paenibacillusterrigena] WP_018756045.1 α-fucosidase [Ruminococcus obeum]WP_005422251.1 α-L-fucosidase [Paenibacillus sp. FSL H7-689] ETT43086.1α-fucosidase [Paenibacillus lactis] WP_007127626.1 α-fucosidase[Bacillus sp. J13] WP_028406965.1 hypothetical protein [Paenibacillusdaejeonensis] WP_020617104.1 hypothetical protein [Clostridium sp KLE1755] WP_021638714.1 α-fucosidase [Clostridium sp. ASBs410]WP_025233568.1 α-fucosidase [Paenibacillus vortex] WP_006211772.1α-L-fucosidase [Paenibacillus sp. FSL R5-808] ETT35249.1 α-fucosidase[Clostridium celerecrescens] KEZ90324.1 α-L-fucosidase [Firmicutesbacterium CAG:94] WP_022336739.1 α-fucosidase [Clostridiales bacteriumVE202-27] WP_025488431.1 α-fucosidase [Paenibacillus pasadenensis]WP_028597616.1 MULTISPECIES: α-fucosidase [Paenibacillus] WP_024629466.1α-fucosidase [Paenibacillus sp. UNC451MF] WP_028551519.1 α-fucosidase[Paenibacillus sp. PAMC 26794] WP_026081066.1 α-fucosidase[Paenibacillus sp. JDR-2] WP_015843379.1 MULTISPECIES: α-fucosidase[Clostridiales] WP_009250084.1 α-fucosidase [Clostridiumsaccharolyticum] WP_013273060.1

In other particular embodiment, the fucosidase is α1,2-fucosidase or aα1,2-transfucosidase, that is those wild type or engineered fucosidasesthat are able to transfer a fucose residue to the 2-position of thegalactose in an acceptor of formula 2, or to the 3-position of thegalactose of the lacto-N-biosyl group an acceptor of formula 1, 1a or1b. In this regard, the preferred compounds of Gly-A obtainable byα1,2-fucosylation of a suitable acceptor are difucosyllactose,lacto-N-fucopentaose I, lacto-N-difuco-hexaose I, F-LNH I, DF-LNH I,F-LST b and FS-LNH.

More preferably, the enzyme having α1,2-fucosidase and/orα1,2-trans-fucosidase activity may be selected from α-L-fucosidasesderived from Thermotoga maritima MSB8, Sulfolobus solfataricus P2,Bifidobacterium bifidum JCM 1254, Bifidobacterium bifidum JCM 1254,Bifidobacterium longum subsp. infantis ATCC 15697, Bifidobacteriumlongum subsp. infantis ATCC 15697, Bifidobacterium longum subsp.Infantis JCM 1222, Bifidobacterium bifidum PRL2010, Bifidobacteriumbifidum S17, Bifidobacterium longum subsp longum JDM 301,Bifidobacterium dentium Bd1, or Lactobacillus casei BL23, etc.

Even more preferably the enzyme having α1,2-fucosidase and/orα1,2-trans-fucosidase activity may be selected from followingα-L-fucosidases as defined according to the following deposit numbers:gil4980806 (Thermotoga maritima MSB8), gil13816464 (Sulfolobussolfataricus P2), gil34451973 (Bifidobacterium bifidum JCM 1254),gil242345155 (Bifidobacterium bifidum, JCM 1254), gil213524647(Bifidobacterium longum subsp. infantis, ATCC 15697), gil213522629(Bifidobacterium longum subsp. infantis ATCC 15697), gil213522799(Bifidobacterium longum subsp. infantis ATCC 15697), gil213524646(Bifidobacterium longum subsp. infantis ATCC 15697), gil320457227(Bifidobacterium longum subsp. infantis JCM 1222), gil320457408(Bifidobacterium longum subsp. infantis JCM 1222), gil320459369(Bifidobacterium longum subsp. infantis JCM 1222), gil320459368(Bifidobacterium longum subsp. infantis JCM 1222), gil310867039(Bifidobacterium bifidum PRL2010), gil310865953 (Bifidobacterium bifidumPRL2010), gil309250672 (Bifidobacterium bifidum S17), gil309251774(Bifidobacterium bifidum S17), gil296182927 (Bifidobacterium longumsubsp longum JDM 301), gil296182928 (Bifidobacterium longum subsp longumJDM 301), gil283103603 (Bifidobacterium dentium Bd1), gil190713109(Lactobacillus casei BL23), gil190713871 (Lactobacillus casei BL23),gil190713978 (Lactobacillus casei BL23), etc., or a sequence exhibitinga sequence identity with one of the above mentioned enzyme sequenceshaving α1,2-fucosidase and/or α1,2-trans-fucosidase activity of at least70%, more preferably at least 80%, equally more preferably at least 85%,even more preferably at least 90% and most preferably at least 95% oreven 97%, 98% or 99% as compared to the entire wild type sequence onamino acid level.

In other embodiment, the glycosidase is a sialidase. Sialidases (EC3.2.1.18), classified in the GH33 family, are retaining enzymes with theability of hydrolysing α-linkage of the terminal sialic acid, mainlythose bound to galactose with α-2,3 or α-2,6 linkage, of varioussialoglycoconjugates. They are found particularly in diverse virusfamilies and bacteria, and also in protozoa, some invertebrates andmammalian. Some bacterial sialidases can be used to scavenge sialicacids from sialylated glycoprotein, glycolipids or other glycoconjugatesfor nutriment for bacterial cell growth.

Although sialidases are characterized by their hydrolytic activity,under appropriate reaction condition they are able to catalyse thetransfer of sialic acid unit to an asialo acceptor by transsialylationreaction giving rise to the formation of sialoglycoconjugates.Sialidases from pathogen bacteria or viruses such as Bacteroidesfragilis, Clostridium species (e.g. C. perfringens), Corynebacteriumdiphteriae, Haemophilus parasuis, Pasteurella multocida, Pseudomonasaeruginosa, Salmonella typhimurium, Streptococcus pneumoniae, Tannerellaforsythia, Vibrio cholerae or Newcastle disease virus and fromnon-pathogen ones such as Actinomyces viscosus, Arthrobacter species orMicromonospora viridifaciens are capable to act as catalyst forsialylation reaction due to their transsialidase activity with α-2,3and/or α-2,6 selectivity. As to the regioselectivity, the ratio betweenthe α-2,3- and α-2,6-linked products varies depending on the enzymesand/or the acceptors. For example sialidases from A. ureafaciens, C.perfringens and V. cholerae have good α-2,6 selectivity, whereas thosefrom S. typhimurium and Newcastle disease virus have good to excellentpreference to form α-2,3 linkage.

Recently, sialidases from Bifidobacterium bifidum and Bifidobacteriumlongum subsp. infantis have been identified, cloned and characterized.These sialidases can cleave and so recognize both α-2,3- andα-2,6-linked sialosides. Sialidases from Bifidobacterium longum subsp.infantis have a consistent preference for α-2,6-linkage whereassialidases from Bifidobacterium bifidum have a consistent preference forα-2,3-linkage.

In order to improve regioselectivity and/or conversion of thetranssialylation reaction the sialidases may be subjected to alterationby various engineering techniques (see above).

In one particular embodiment, the sialidase is an α2,3-sialidase or anα2,3-transsialidase, that is those wild type or engineered sialidasesthat are able to transfer a sialyl residue to the 3-position of thegalactose in an acceptor of formula 2, or to the 3-position of thegalactose in a terminal lacto-N-biosyl group in an acceptor of formula1, 1a or 1b. In this regard, the preferred compounds of Gly-A obtainableby α2,3-sialylation of a suitable acceptor areNeu5Acα2-3Galβ1-4(Fucα1-3)Glc (3-O-fucosyl-3′-O-sialyl-lactose),Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc (LST a),Neu5Acα2-3Galβ13-4GlcNAcβ1-3Galβ1-4Glc,Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FLST a),Neu5Acα2-3Galβ1-4(Fucα1-3)GlcNAcβ1-3Galβ1-4Glc,Neu5Acα2-3Galβ1-3GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Neu5Acα2-3Galβ1-3(Fucα1-4)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc,Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4Glc (DSLNT),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)(Fucα1-4)GlcNAcβ1-3Galβ1-4Glc (FDSLNT I),Neu5Acα2-3Galβ1-3(Neu5Acα2-6)GlcNAcβ1-3Galβ1-4(Fucα1-3)Glc (FDSLNT II).

The α2,3-transsialidase suitable in the processes of this invention ispreferably the α2,3-transsialidase from Trypanosoma cruzi (TcTS).However, the α2,3-transsialidases from other microorganisms, such as T.rangeli, T. brucei gambiense, T. brucei rhodesiense, T. brucei brucei,T. congolense and Corynebacterium diphtheriae as described in WO2012/156898, as well as the α2,3-transsialidases from Salmonellatyphimurium, Bacteroides fragilis, Newcastle disease virus and Vibriocholera, can be used. Moreover, other α2,3-transsialidases can also beused which have at least 60%, preferably at least 70%, more preferablyat least 80%, particularly at least 90%, identity with theα2,3-transsialidase from T. cruzi. Also preferably, theα2,3-transsialidase may be selected from sialidases or transsialidasesas defined according to the following deposit numbers: gil213524659(Bifidobacterium longum subsp. infantis ATCC 15697), gil213523006Bifidobacterium longum subsp. infantis ATCC 15697), gil309252191(Bifidobacterium bifidum S17), gil309252190 (Bifidobacterium bifidumS17), gil310867437 (Bifidobacterium bifidum PRL2010), gil310867438(Bifidobacterium bifidum PRL2010), gil224283484 (Bifidobacterium bifidumNCIMB 41171), gil224283485 (Bifidobacterium bifidum NCIMB 41171),gil334283443 (Bifidobacterium bifidum JCM1254), gil47252690 (T. cruzi),gil432485 (T. cruzi).

In other particular embodiment, the sialidase is an α2,6-sialidase or anα2,6-transsialidase, that is those wild type or engineered sialidasesthat are able to transfer a sialyl residue to the 6-position of thegalactose in a terminal N-acetyllactosaminyl group in an acceptor offormula 1, 1a or 1b, or to the 6-position of the N-acetylglucosamine ina, preferably terminal, lacto-N-biosyl group in an acceptor of formula1, 1a or 1b. In this regard, the preferred compounds of Gly-A obtainableby α2,6-sialylation of a suitable acceptor are LST c, FLST c, SLNH,SLNnH-I, SLNnH-II, FSLNH, FSLNH-III, FSLNnH-I, FSLNnH-II, DFSLNH-I,DFSLNnH, DSLNH-I, DSLNnH, DSLNnH, FDSLNH-III, FDSLNnH, FDSLNnH, TSLNH.

The α2,6-transsialidase suitable in the process of this invention can beany wild type enzyme having α2,6-transsialidase activity, such as anα2,6-sialyl transferase from Photobacterium damselae JT0160 (U.S. Pat.Nos. 5,827,714, 6,255,094, Yamamoto et al. J. Biochem. 123, 94 (1998)),Photobacterium sp. JT-ISH-224 (U.S. Pat. Nos. 7,993,875, 8,187,838,Tsukamoto et al. J. Biochem. 143, 187 (2008)) P. leiognathi JT-SHIZ-145(U.S. Pat. Nos. 8,187,853, 8,372,617, Yamamoto et al. Glycobiology 17,1167 (2007)) or P. leiognathi JT-SHIZ-119 (US 2012/184016, Mine et al.Glycobiology 20, 158 (2010)). The preferred wild type α2,6-sialyltransferases with a substantially identical amino acid sequence with SEQID No. 1, that is having at least about 60 percent sequence identity(determined by BLAST) with SEQ ID No. 1, are listed in Table 2.

TABLE 2 Description Identity Accession Number α2,6-sialyl transferase100% BAI49484.1 [Photobacterium leiognathi] α2,6-sialyl transferase  96%BAF91416.1 [Photobacterium leiognathi] Chain A, crystal structure ofsialyl  70% 4R9V_A transferase from Photobacterium damselae, residues113-497 sialyl transferase 0160  68% WP_005298232.1 [Photobacteriumdamselae] Chain A, crystal structure of  67% 4R83_A sialyl transferasefrom Photobacterium damselae sialyl transferase 0160  66% BAA25316.1[Photobacterium damselae]

Preferably, α2,6-sialyl transferases with a substantially identicalamino acid sequence with SEQ ID No. 1 that can be mutated to have anα2,6-transsialidase activity with improved regioselectivity, are thesialyl transferase from P. leiognathi JT-SHIZ-119 or its A2-15 truncatedvariant, the sialyl transferase from P. leiognathi JT-SHIZ-145 or itsA2-15 truncated variant, or the sialyl transferase from P. damselaeJT0160 or its A2-15 truncated variant, more preferably the sialyltransferase from P. leiognathi JT-SHIZ-119 or its A2-15 truncatedvariant. Especially preferred α2,6-transsialidases obtained by enzymeengineering are disclosed in WO 2016/199069.

In other embodiment, the glycosidase is a lacto-N-biosidase ortrans-lacto-N-biosidase, that is those wild type or engineeredlacto-N-biosidases that are able to transfer a lacto-N-biosyl residue tothe 3-position of the galactose of lactose, to the 3-position of thegalactose in a terminal N-acetyllactosaminyl group in an acceptor offormula 1, 1a or 1b, or to the 3-position of the galactose in a terminallacto-N-biosyl group in an acceptor of formula 1, 1a or 1b. In thisregard, the preferred compounds of Gly-A obtainable byβ1,3-lacto-N-biosylation of a suitable acceptor are lacto-N-hexaose(LNH, Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc),para-lacto-N-hexaose (para-LNH,Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc), para-lacto-N-hexaose II(para-LNH II, Galβ1-3GlcNAcβ1-3Galβ1-3GlcNAcβ1-3Galβ1-4Glc),lacto-N-octaose (LNO,Galβ1-3GlcNAcβ1-3(Galβ1-4GlcNAcβ1-3Galβ11-4GlcNAcβ1-6)Galβ1-4Glc),iso-lacto-N-octaose (iso-LNO,Galβ1-3GlcNAcβ1-3(Galβ11-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc),para-lacto-N-octaose (para-LNO,Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc).

Enzymes having a lacto-N-biosidase or trans-lacto-N-biosidase activityare preferably selected from a lacto-N-biosidase ortrans-lacto-N-biosidase (EC 3.2.1.140) as classified according to theGH20 family. Lacto-N-biosidases typically proceed through a retainingmechanism. Only two lacto-N-biosidases from Streptomyces andBifidobacterium bifidum have been described and characterized up to now,which may be utilized in the present invention as a lacto-N-biosidase ortrans-lacto-N-biosidase (see Sano et al., Proc. Natl. Acad. Sci. USA,89, 8512 (1992); Sano et al., J. Biol. Chem. 268, 18560 (1993); Wada etal., Appl. Environ. Microbiol. 74, 3996 (2008)). Lacto-N-biosidasesspecifically hydrolyse the terminal lacto-N-biosyl residue(β-D-Gal-(1→3)-D-GlcNAc) from the non-reducing end of oligosaccharideswith the structure β-D-Gal-(1→3)-β-D-GlcNAc-(1→3)-β-D-Gal-(1→R). Wada etal. (supra) and Murata et al. (Glycoconj. J. 16, 189 (1999)) alsodemonstrated the ability of the lacto-N-biosidase from Bifidobacteriumbifidum and Aureobacterium sp. L-101, respectively, to catalyse thetransglycosylation by incubating donor substrates (such aslacto-N-tetraose and pNP-β-LNB) with acceptors (such as various1-alkanols and lactose).

Even more preferably, the at least one enzyme having a lacto-N-biosidaseor trans-lacto-N-biosidase activity may be selected fromlacto-N-biosidases or trans-lacto-N-biosidases derived fromBifidobacterium bifidum JCM1254, Bifidobacterium bifidum PRL2010,Bifidobacterium bifidum NCIMB 41171, Aureobacterium sp. L-101 orStreptomyces sp., etc.

Even more preferably the at least one enzyme having a lacto-N-biosidaseor trans-lacto-N-biosidase activity may be selected fromlacto-N-biosidases or trans-lacto-N-biosidases as defined according tothe following deposit numbers: gil167369738 (Bifidobacterium bifidumJCM1254), gil4096812 (Streptomyces sp.), gil310867103 (Bifidobacteriumbifidum PRL2010), gil313140985 (Bifidobacterium bifidum NCIMB 41171),etc., or a sequence exhibiting a sequence identity with one of the abovementioned enzyme sequences having a lacto-N-biosidase ortrans-lacto-N-biosidase activity of at least 70%, more preferably atleast 80%, equally more preferably at least 85%, even more preferably atleast 90% and most preferably at least 95% or even 97%, 98% or 99% ascompared to the entire wild type sequence on amino acid level.

In other embodiment, the glycosidase is an N-acetyllactosaminidase ortrans-N-acetyllactosaminidase, that is those wild type or engineeredN-acetyllactosaminidases that are able to transfer aN-acetyllactosaminyl residue to the 3-position of the galactose oflactose, or to the 3-position of the galactose in a terminalN-acetyllactosaminyl group in an acceptor of formula 1, 1a or 1b. Inthis regard, the preferred compounds of Gly-A obtainable byβ1,3-N-acetyllactosaminylation of a suitable acceptor arelacto-N-neohexaose (LNnH,Galβ1-4GlcNAcβ1-3(Galβ1-4GlcNAcβ1-6)Galβ1-4Glc), para-lacto-N-neohexaose(para-LNnH, Galβ1-4GlcNAcβ1-3Galβ1-4GlcNAcβ1-3Galβ1-4Glc),lacto-N-neooctaose (LNnO,Galβ1-4GlcNAcβ1-3(Galβ1-3GlcNAcβ1-3Galβ1-4GlcNAcβ1-6)Galβ1-4Glc).

Enzymes having a N-acetyllactosaminidase ortrans-N-acetyllactosaminidase activity are preferably selected from aN-acetyllactosaminidase or trans-N-acetyllactosaminidase as described inthe following, e.g. lacto-N-biosidases (EC 3.2.1.140) as classifiedaccording to the GH20 family. Particularly preferably, chitinase frombacillus circulans, more preferably chitinase A1 from Bacillus CirculansWL-12 as deposited under gil142688, may be used as aN-acetyllactosaminidase or trans-N-acetyllactosaminidase, or a sequenceexhibiting a sequence identity with one of the above mentioned enzymesequences having a N-acetyllactosaminidase ortrans-N-acetyllactosaminidase activity of at least 70%, more preferablyat least 80%, equally more preferably at least 85%, even more preferablyat least 90% and most preferably at least 95% or even 97%, 98% or 99% ascompared to the entire wild type sequence on amino acid level. Notably,Shoda et al. showed that chitinase A1 from B. Circulans WL-12 is able totransfer N-acetyllactosamine with a β-1,6 glycosidic linkage using1,2-oxazoline derivative of N-acetyllactosamine (Cellulose, 13, 477(2006)).

In one embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-tetraose (LNT), Gly-A is lacto-N-fucopentaose II (LNFP-II), andthe glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-neotetraose (LNnT), Gly-A is lacto-N-fucopentaose III(LNFP-III), and the glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-fucopentaose I (LNFP-I), Gly-A is lacto-N-difucohexaose I(LNDFH-I), and the glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-fucopentaose V (LNFP-V), Gly-A is lacto-N-difucohexaose II(LNDFH-II), and the glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-fucopentaose VI (LNFP-VI), Gly-A is lacto-N-difucohexaose I(LNDFH-III), and the glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A is2′-O-fucosyllactose (2′-FL), Gly-A is difucosyllactose (DFL), and theglycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 2′-O-fucosyllactose (2′-FL), A is3-O-fucosyllactose (3-FL), Gly-A is difucosyllactose (DFL), and theglycosidase is an α1,2-(trans)fucosidase.

In other embodiment, Gly-B is 3′-O-sialyllactose (3′-SL), A is3-O-fucosyllactose (3-FL), Gly-A is 3-O-fucosyl-3′-O-sialyllactose(FSL), and the glycosidase is an α2,3-transsialidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A is3′-O-sialyllactose (3′-SL), Gly-A is 3-O-fucosyl-3′-O-sialyllactose(FSL), and the glycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 3′-O-sialyllactose (3′-SL), A islacto-N-tetraose (LNT), Gly-A is LST a, and the glycosidase is anα2,3-transsialidase.

In other embodiment, Gly-B is 3′-O-sialyllactose (3′-SL), A islacto-N-fucopentaose II (LNFP-II), Gly-A is FLST a, and the glycosidaseis an α2,3-transsialidase.

In other embodiment, Gly-B is 6′-O-sialyllactose (6′-SL), A islacto-N-neotetraose (LNnT), Gly-A is LST c, and the glycosidase is anα2,6-(trans)sialidase.

In other embodiment, Gly-B is 6′-O-sialyllactose (6′-SL), A islacto-N-fucopentaose VI (LNFP-VI), Gly-A is FLST c, and the glycosidaseis an α2,6-(trans)sialidase.

In other embodiment, Gly-B is 3-O-fucosyllactose (3-FL), A ispara-lacto-N-neohexaose (pLNnH), Gly-A is a fucosylated pLNnH, and theglycosidase is an α1,3/4-(trans)fucosidase.

In other embodiment, Gly-B is 6′-O-sialyllactose (6′-SL), A ispara-lacto-N-neohexaose (pLNnH), Gly-A is a sialylated pLNnH, and theglycosidase is an α2,6-(trans)sialidase.

Also in a preferred embodiment, the separation factor of disaccharide Bover a tri- or higher oligosaccharide Gly-A, A or Gly-B is more than 5,preferably more than 10, more preferably more than 25, even morepreferably more than 100. Especially, the separation factor of lactoseover a human milk oligosaccharide Gly-A is more than 10, preferably morethan 25, more preferably more than 50, even more preferably more than100.

Yet preferably, the separation factor of disaccharide B over atrisaccharide A or Gly-B is more than 5, preferably more than 10, morepreferably more than 25. Especially, the separation factor of lactoseover 3′-SL or 6′-SL is more than 20, preferably more than 50.

Yet preferably, the separation factor of disaccharide B over atetrasaccharide A or Gly-A is more than 25, preferably more than 50,more preferably more than 100. Especially, the separation factor oflactose over LNT or LNnT is more than 30, more preferably more than 50.

Yet preferably, the separation factor of disaccharide B over a penta- orhexasaccharide A or Gly-A is more than 100. Especially, the separationfactor of lactose over LNFP-I, LNFP-II, LNFP-III, LNFP-V, LNFP-VI, LSTa, LST c, FLST a, FLST c, LNDFH-I, LNDFH-II or LNDFH-III is more than150, more preferably more than 250.

The method of the invention can be conducted under conditions used forconventional nanofiltration with tangential flow or cross-flowfiltration with positive pressure compared to permeate side followed by,optionally, diafiltration where both operations could be performed in abatch mode or preferably in continuous mode. The optional diafiltrationis conducted by adding pure water to the retentate after thenanofiltration step disclosed above and continuing the filtrationprocess. Conducting diafiltration helps to remove the continuouslyforming lactose from the reactor more efficiently, therefore shifts theenzymatic equilibrium towards the product formation.

The pH of the feed solution applied for the NF separation according tothe present invention is, preferably, not higher than 7, more preferablybetween 3 and 7, even more preferably around 4 and 5. A low pH mayadversely influence the membrane and the solute properties.Nevertheless, the pH shall be in a range under which the(trans)glycosidase is capable of performing the transfer of the Glymoiety from the donor to the acceptor.

The convenient temperature range applied is between 10 and 60° C. Highertemperature provides a higher flux and thus accelerates the process. Themembrane is expected to be more open for flow-through at highertemperatures, however this doesn't change the separation factorssignificantly. On the other hand, since the enzymatic reaction and thecontinuous removal of the lactose take place parallelly, the preferredtemperature range for conducting the nanofiltration separation accordingto the invention is that under which the (trans)glycosidase is capableof performing the transfer of the Gly moiety from the donor to theacceptor. It is, in general, 20-40° C., however in case of thermostableenzyme the temperature may be as high as 50° C.

A preferred applied pressure in the nanofiltration separation is about2-50 bar, such as 10-40 bar, the higher the pressure the higher theflux.

In the first aspect of the invention, preferably, the enzymatic reactionis conducted in an enzymatic membrane reactor (see e.g. Luo et al.(Biores. Technol. 166, 9 (2014)) composed of the membrane defined, sothat compounds Gly-B (donor), A (acceptor) and Gly-A (product) remain inthe reactor (“retentate”) since they are all tri- or higheroligosaccharides, while compound B as the leaving group of the donor andbeing a disaccharide can penetrate through the membrane to the permeatefraction. In such membrane reactor, the equilibrium of the enzymaticreaction can be shifted towards the product formation, thus a betterconversion is achievable compared to the conventional reactors.

In the second aspect of the invention, both conventional and membranereactors can be applied, however the enzymatic mixture comprisingcompounds Gly-B (donor), A (acceptor), Gly-A (product) and B, afterreaching a certain conversion or equilibrium, is contacted with thenanofiltration membrane under diafiltration mode using pure water. Indoing so, the enzymatic reaction keeps going because the disaccharide Bis continuously removed through the membrane to the permeate fractionand its reduced concentration shifts the equilibrium towards itsproduction together with the production of Gly-A which remains in theretentate.

In the final step of the method of invention, the mixture of A, Gly-Aand Gly-B (and optionally B if it remains in a small amount) in solidform can then be isolated from the aqueous solution obtained asretentate after UF/DF in a conventional manner, first separating theenzyme from the retentate followed by e.g. by evaporation, spray-dryingdrying, or lyophilisation. Gly-A can be isolated in pure form frommixture of A, Gly-A and Gly-B (and optionally B) by conventionalseparation method such as gel chromatography, reversed-phasechromatography, ion exchange chromatography or ligand exchangechromatography.

EXAMPLES Example 1—Determination of the Rejection Factor of a Substanceon a Membrane

The NaCl and MgSO₄ rejection on a membrane is determined as follows: ina membrane filtration system, a NaCl (0.1%) or a MgSO₄ (0.2%) solutionis circulated across the selected membrane sheet (for Tami: tubularmodule) while the permeate stream is circulated back into the feed tank.The system is equilibrated at 10 bars and 25° C. for 10 minutes beforetaking samples from the permeate and retentate. The rejection factor iscalculated from the measured conductivity of the samples:(1−κ_(p)/κ_(r))·100, wherein κ_(p) is the conductivity of MgSO₄ in thepermeate and Kr is the conductivity of NaCl or MgSO₄ in the retentate.

NaCl rej. factor MgSO₄ rej. factor supplier lab. supplier lab. membraneactive layer MWCO spec. measurement spec. measurement Triseppiperazine-PA 1000-3500 — 10% 80% 81-89%    UA60 GE GH PA 2500 — 81% —76% NTR-7450 sulph. PES 600-800 50% 56% — 32%

A carbohydrate rejection factor is determined in a similar way with thedifference that the rejection factor is calculated from theconcentration of the samples (determined by HPLC): (1−C_(p)/C_(r))·100,wherein C_(p) is the concentration of the carbohydrate in the permeateand C_(r) is the concentration of the carbohydrate in the retentate.

Example 2—LST c Production Catalysed by an α2,6-Transsialidase withContinuous Lactose Removal

6′-SL Na-salt (80.0 g) and LNnT (60.0 g) were dissolved in deionizedwater (860 g), and the pH was adjusted to 5.0 with few drops of aceticacid. α2,6-Transsialidase (A218Y-N222R-G349S-S412P-D451K mutant of P.leiognathi JT-SHIZ-119 sialyl transferase truncated by its signalpeptide (Δ2-15), the positions of mutations are according to SEQ ID No.1, see WO 2016/199069) was added in two portions (50 mg at the start and100 mg after 4 hrs) and the obtained solution was agitated at ambienttemperature for 21 hrs to give an equilibrated mixture of 6′-SL, LNnT,LST c and lactose with ca. 38% conversion. The obtained solution wassubjected to diafiltration (DF) in the cross-flow MMS SW18 filtrationsystem with installed Trisep UA60 membrane (piperazine PA, MWCO1000-3500 Da, measured MgSO₄ rejection is 89%, spiral-wound, size 1812,area 0.23 m²) at p=15-20 bar and T=25-30° C. with DF water (flow rate inthe range of 3-4.5 l/h, matching approximately the permeate flow rate).During the process, additional amount of enzyme was added periodicallyby small portions (7×50 mg, 300 mg in total). pH was measuredperiodically and adjusted if necessary by adding small amount of sodiumacetate to keep it in the range of 4.5-5.5. After consumption of 25 l ofwater, the permeate collection was paused overnight while keeping thereaction mixture circulating slowly in the system at low temperature(+8° C.). Next day DF continued with another 25 l of water under thesame conditions. The obtained retentate was pumped out from the system(746 g) and the remaining dead volume was removed by washing with twoportions of water (2×350 ml). The obtained diluted retentate (1448 g)was heated up to 85° C. in 30 min. The obtained suspension was allowedto cool, treated with charcoal, filtered and the filtrate wasconcentrated and freeze-dried to give 83.54 g of a colourless solid.Analytical samples were periodically taken and analysed by HPLC. Theobtained amounts and conversion are summarized in the table below.

Volume/mass 6′-SL lactose LST c LNnT conversion Initial 950 ml 80 g — —60 g MW (Da) 655.5 (Na-salt) 342 997 707 t = 2 min (mmol) 1000 ml 125 —— 82.38 t = 21 h (before DF, mmol) 1000 ml 97.8 31.1 31.1 51.26 37.9%permeate 1, 0-25 l (mmol) 25 l 1.78 41.41 2.63 4.44 permeate 2, 25-50 l(mmol) 25 l 0.89 9.82 1.72 1.94 combined permeate (mmol) 50 l 2.68 51.234.35 6.38 final diluted retentate after 1448 g 34.46 0 46.57 10.30 DFwith 50 l water (mmol) permeate + retentate (mmol) 37.14 51.23 50.9216.68 75.4%

Example 3

Flat sheet membranes (d=20 cm, active membrane area 280 cm² for eachsheet) were installed into a cross-flow flat sheet cell of the MMS SW18membrane filtration system. Pure water was equilibrated at 10 bars and23-25° C. with constant cross-flow (300 l/h) for at least 10 min. Thensmall portion (5-30 ml) of permeate fractions were collected and exactmass or volume was measured. Flux was calculated according to thefollowing formula: F=V/(t·A) where V is the collected permeate volume inlitres, t is the time required to collect the measured volume in hoursand A is the membrane area in m².

The following pure water flux values were measured:

membrane active layer MWCO flux (l/m²h) Trisep UA60 piperazine-PA1000-3500 100.8 GE GH PA 2500 17  NTR-7450 sulph. PES 600-800  99.6

Then, for the Trisep UA60 and Nitto-Denko NTR-7450 membranes, water wasreplaced by a feed solution which was prepared as follows: crude LNnTsolid sample was obtained from fermentation broth after cell removal byUF (15 kDa), NF (150-300 Da) with diafiltration, decolouration withactivated charcoal and freeze-drying. The obtained solid contained LNnT(54.6%), lactose (9.86%), lacto-N-triose II (7.32%) and pLNnH (8.67%,all by weight), from which 41 g was dissolved in 2050 g of water,obtaining a solution having a pH of 5.71 and conductivity of 0.825mS/cm. The flux of the feed solution was measured under the sameconditions.

Then the membranes were washed with pure water (cleaning in place,CIP1), and water flux was re-measured.

Following this, the membranes were washed with an aqueous cleaningsolution containing 0.1% sodium dodecyl sulphate, 0.5% EDTA and 0.5%sodium tripolyphosphate (cleaning in place, CIP2, 30 min, 5 bar, 20-25°C.), and water flux was remeasured.

The data show that the NTR-7450 membrane is more prone to being fouledthan Trisep UA60 when a pre-treated oligosaccharide solution obtainedafter fermentation is applied. Furthermore, while pure water washingregenerated the Trisep UA60 membrane to reach 85% of the original waterflux, it was inefficient to do so for the NTR-7450 membrane. Inaddition, whereas a detergent containing cleaning solution completelycleaned the Trisep UA60 membrane, the NTR-4750 membrane was regeneratedonly partially.

flux (l/m²h) Trisep UA60 NTR-7450 initial water flux 100.8 99.6 fluxwith feed solution  55.1 30.3 water flux after CIP1  85.4 23.9 afterCIP1 relative to initial  85% 24% water flux after CIP2 119   71.3 afterCIP2 relative to initial 118% 72%

1. A method for producing a compound Gly-A by glycosylating acceptor Awith a donor Gly-B under catalysis of a glycosidase capable oftransferring a Gly moiety from the donor to the acceptor thereby forminga mixture containing Gly-A, A, Gly-B and B, comprising: a) contactingsaid mixture with a nanofiltration membrane with a molecular weightcut-off (MWCO) of 600-3500 Da allowing at least a part of compound B topass and ensuring the retention of compounds Gly-A, A and Gly-B, whereinthe membrane comprises an active layer of polyamide, and a MgSO₄rejection of 50-90%, and b) collecting a retentate enriched in compoundGly-A, wherein A means a tri- or higher oligosaccharide, B means adisaccharide, Gly means a glycosyl moiety, Gly-A means compound Aglycosylated with the Gly moiety, and Gly-B means compound Bglycosylated with the Gly moiety, provided that compounds A and Gly-Bare not identical.
 2. A method for improving conversion of productformation of compound Gly-A in an enzymatic reaction wherein acceptor Ais glycosylated with a donor Gly-B under catalysis of a glycosidasecapable of transferring a Gly moiety from the donor to the acceptorthereby forming a mixture containing Gly-A, A, Gly-B and B, comprising:a) contacting said mixture, under diafiltration condition, with ananofiltration membrane with a molecular weight cut-off (MWCO) of600-3500 Da allowing at least a part of compound B to pass and ensuringthe retention of compounds Gly-A, A and Gly-B, wherein the membranecomprises an active layer of polyamide, and a MgSO₄ rejection of 50-90%,and b) collecting a retentate enriched in compound Gly-A, wherein Ameans a tri- or higher oligosaccharide, B means a disaccharide, Glymeans a glycosyl moiety, Gly-A means compound A glycosylated with theGly moiety, and Gly-B means compound B glycosylated with the Gly moiety,provided that compounds A and Gly-B are not identical.
 3. The methodaccording to claim 1, wherein the membrane comprises a NaCl rejectionlower than the MgSO₄ rejection.
 4. The method according to claim 3,wherein the NaCl rejection of the membrane is not more than 30%.
 5. Themethod according to claim 1, wherein the method comprises diafiltration.6. The method according to claim 1, wherein the polyamide nanofiltrationmembrane is a thin-film composite (TFC) membrane.
 7. The methodaccording to claim 1, wherein the pure water flux of the membrane is atleast 50 l/m²h.
 8. The method according to claim 1, wherein thepolyamide nanofiltration membrane is a piperazine/based polyamidemembrane.
 9. The method according to claim 1, wherein the disaccharideis lactose.
 10. The method according to claim 1, wherein the glycosylmoiety is fucosyl and the glycosidase is a fucosidase or atransfucosidase, or the glycosyl moiety is sialyl and the glycosidase isa sialidase or a transsialidase.
 11. The method according to claim 1,wherein Gly-B is 3-O-fucosyllactose (3-FL), A is lacto-N-tetraose (LNT),Gly-A is lacto-N-fucopentaose II (LNFP-II), and the glycosidase is anα1,3/4-(trans)fucosidase, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-neotetraose (LNnT), Gly-A is lacto-N-fucopentaose III(LNFP-III), and the glycosidase is an α1,3/4-(trans)fucosidase, Gly-B is3-O-fucosyllactose (3-FL), A is lacto-N-fucopentaose I (LNFP-I), Gly-Ais lacto-N-difucohexaose I (LNDFH-I), and the glycosidase is anα1,3/4-(trans)fucosidase, Gly-B is 3-O-fucosyllactose (3-FL), A is2′-O-fucosyllactose (2′-FL), Gly-A is difucosyllactose (DFL), and theglycosidase is an α1,3/4-(trans)fucosidase, Gly-B is 2′-O-fucosyllactose(2′-FL), A is 3-O-fucosyllactose (3-FL), Gly-A is difucosyllactose(DFL), and the glycosidase is an α1,2-(trans)fucosidase, Gly-B is3′-O-sialyllactose (3′-SL), A is 3-O-fucosyllactose (3-FL), Gly-A is3-O-fucosyl-3′-O-sialyllactose (FSL), and the glycosidase is anα2,3-transsialidase, Gly-B is 3-O-fucosyllactose (3-FL), A is3′-O-sialyllactose (3′-SL), Gly-A is 3-O-fucosyl-3′-O-sialyllactose(FSL), and the glycosidase is an α1,3/4-(trans)fucosidase, Gly-B is3′-O-sialyllactose (3′-SL), A is lacto-N-tetraose (LNT), Gly-A is LST a,and the glycosidase is an α2,3-transsialidase, Gly-B is6′-O-sialyllactose (6′-SL), A is lacto-N-neotetraose (LNnT), Gly-A isLST c, and the glycosidase is an α2,6-(trans)sialidase, Gly-B is3-O-fucosyllactose (3-FL), A is para-lacto-N-neohexaose (pLNnH), Gly-Ais a fucosylated pLNnH, and the glycosidase is anα1,3/4-(trans)fucosidase, or Gly-B is 6′-O-sialyllactose (6′-SL), A ispara-lacto-N-neohexaose (pLNnH), Gly-A is a sialylated pLNnH, and theglycosidase is an α2,6-(trans)sialidase.
 12. The method according toclaim 2, wherein the membrane comprises a NaCl rejection lower than theMgSO₄ rejection.
 13. The method according to claim 12, wherein the NaClrejection of the membrane is not more than 30%.
 14. The method accordingto claim 2, wherein the method comprises diafiltration.
 15. The methodaccording to claim 2, wherein the polyamide nanofiltration membrane is athin-film composite (TFC) membrane.
 16. The method according to claim 2,wherein the pure water flux of the membrane is at least 50 l/m²h. 17.The method according to claim 2, wherein the polyamide nanofiltrationmembrane is a piperazine/based polyamide membrane.
 18. The methodaccording to claim 2, wherein the disaccharide is lactose.
 19. Themethod according to claim 2, wherein the glycosyl moiety is fucosyl andthe glycosidase is a fucosidase or a transfucosidase, or the glycosylmoiety is sialyl and the glycosidase is a sialidase or a transsialidase.20. The method according to claim 2, wherein Gly-B is 3-O-fucosyllactose(3-FL), A is lacto-N-tetraose (LNT), Gly-A is lacto-N-fucopentaose II(LNFP-II), and the glycosidase is an α1,3/4-(trans)fucosidase, Gly-B is3-O-fucosyllactose (3-FL), A is lacto-N-neotetraose (LNnT), Gly-A islacto-N-fucopentaose III (LNFP-III), and the glycosidase is anα1,3/4-(trans)fucosidase, Gly-B is 3-O-fucosyllactose (3-FL), A islacto-N-fucopentaose I (LNFP-I), Gly-A is lacto-N-difucohexaose I(LNDFH-I), and the glycosidase is an α1,3/4-(trans)fucosidase, Gly-B is3-O-fucosyllactose (3-FL), A is 2′-O-fucosyllactose (2′-FL), Gly-A isdifucosyllactose (DFL), and the glycosidase is anα1,3/4-(trans)fucosidase, Gly-B is 2′-O-fucosyllactose (2′-FL), A is3-O-fucosyllactose (3-FL), Gly-A is difucosyllactose (DFL), and theglycosidase is an α1,2-(trans)fucosidase, Gly-B is 3′-O-sialyllactose(3′-SL), A is 3-O-fucosyllactose (3-FL), Gly-A is3-O-fucosyl-3′-O-sialyllactose (FSL), and the glycosidase is anα2,3-transsialidase, Gly-B is 3-O-fucosyllactose (3-FL), A is3′-O-sialyllactose (3′-SL), Gly-A is 3-O-fucosyl-3′-O-sialyllactose(FSL), and the glycosidase is an α1,3/4-(trans)fucosidase, Gly-B is3′-O-sialyllactose (3′-SL), A is lacto-N-tetraose (LNT), Gly-A is LST a,and the glycosidase is an α2,3-transsialidase, Gly-B is6′-O-sialyllactose (6′-SL), A is lacto-N-neotetraose (LNnT), Gly-A isLST c, and the glycosidase is an α2,6-(trans)sialidase, Gly-B is3-O-fucosyllactose (3-FL), A is para-lacto-N-neohexaose (pLNnH), Gly-Ais a fucosylated pLNnH, and the glycosidase is anα1,3/4-(trans)fucosidase, or Gly-B is 6′-O-sialyllactose (6′-SL), A ispara-lacto-N-neohexaose (pLNnH), Gly-A is a sialylated pLNnH, and theglycosidase is an α2,6-(trans)sialidase.